We are applying proteomic methodology to unresolved problems in neuropathologic diseases. In the past year, progress has been made in studies on the structure of the postsynaptic density and its remodeling by drugs used in the treatment of mood disorders (in progress), and on protein complexes implicated by genomic studies of schizophrenia (in progress). A new project has been initiated regarding synaptic protein changes accompanying long term depression (LTD). Increasing evidence supports the hypothesis that bipolar disorder arises from abnormalities in cellular plasticity cascades, leading to aberrant information processing in synapses and circuits. In the context of this hypothesis, mood stabilizers such as lithium and valproic acid are thought to exert their therapeutic effects via actions on systems involved in synaptic plasticity. The postsynaptic density (PSD) is an elaborate cytoskeletal and signaling complex that provides anchors for synaptic proteins close to the region of presynaptic neurotransmitter release, and therefore mediates signaling in a host of divergent signal transduction pathways. Collaborative studies with NINDS have focused on defining the composition of the post-synaptic density complex. Toward that objective, we chose thirty two signature peptides observed consistently in LC/MS/MS analyses, representing the C-and N-terminal regions from 16 prominent proteins isolated with the PSD signaling complex. Synthetic genes are being synthesized to enable generation of stable isotope labeled internal standard proteins that will exhibit proteolytic properties of full-length proteins. These will be used for absolute quantification by multiple reaction monitoring using triple quadrupole mass spectrometry to allow stoichiometric analyses of PSD component proteins. The goal of a second PSD project is to understand the temporal and spatial dynamics of the PSD in the treatment of mood disorders. It is our contention that in addition to manipulating key candidate molecules these studies are critical to elucidate the mechanisms of synaptic regulation and of mood stabilizer action. Previous studies have demonstrated that multiple signaling molecules, including AMPA and NMDA glutamate receptor subunits, and downstream signaling molecules such as glycogen synthase kinase-3 (GSK-3), are regulated by mood stabilizers such as lithium and valproic acid. We have isolated hippocampal PSDs from groups of rats treated chronically with lithium, valproate, or controls. Peptide mixtures from the tryptically digested PSDs, have been separated by ion exchange chromatography prior to LC/MS/MS analysis on a high resolution Orbitrap mass analyzer. Data files from the resulting 324 analyses are being processed using a variety of bioinformatic tools to identify and quantify the consequences of chronic treatment. In preliminary analysis, 512 proteins were identified and quantified (more than two peptides per protein) with very high confidence in all three conditions. Relative quantification data based in ion current (DBParser) and spectral counting (MassSieve) shows that PSD scaffolding proteins levels remain relatively unchanged in valproate and lithium treated animals. The same is true with major glutamate receptors found in PSD with notable exception of GRM2, GRM3, GRM7 and GRIA4. Proteins belonging to ontological groups kinase and phosphatase, voltage dependent calcium channels, cell adhesion, GABA receptors, and G-protein regulators, show significant changes in abundance ratios between different conditions. Statistical and network analyses are being refined prior to publication. The ErbB4 protein signaling complex has been implicated by genomic studies on schizophrenia . We are continuing a project to identify, characterize and validate proteins associated with this receptor tyrosine-protein kinase. Antibodies to ErbB4 have been covalently linked to magnetic beads. These were used to isolate and characterize human ErbB4 from mouse heart in animals that are genetically rescued from embryonic lethality by transgenic expression of human ErbB4 in the heart (ErbB4-/- HER4heart. The same monoclonal antibodies are being used to immuno-capture mouse brain ErbB4. Because there is relatively low expression of this protein, the use of ErbB4-/- is required for immuno-precipitation control. Comparative LC/MS/MS proteomic studies are in progress using newly developed on-bead digestion procedures developed for this project (MH000274-35). Caspase-3 is a cysteine protease best known as an executioner protease in apoptosis. Recent studies suggest that caspase-3 plays an important non-apoptotic function in synapses as well. In hippocampal neurons, caspase-3 activity is specifically required for NMDA receptor-dependent LTD, but not for LTP. More importantly, activation of casapse-3 in LTD promotes AMPA receptor endocytosis instead of causing cell death. In a new study, we propose to address how caspase-3 induces AMPA receptor endocytosis in LTD by using mass spectrometry to identify substrates of caspase-3 cleaved in LTD. We are applying a method developed by Mahrus et al to selectively capture emergent peptides derived from proteolytic cleavage upon NMDA receptor activation. Briefly, the unblocked protein N-termini produced from protease cleavage event are labeled by a biotinylated peptide tag using an engineered enzyme subtiligase. After tryptic digestion, the labeled N-terminal peptides will be enriched by immobilized avidin, released by TEV protease, and analyzed by mass spectrometry.